The circRNA hsa-circ-0013561 regulates head and neck squamous cell carcinoma development via the miR-7-5p/PDK3 axis

Background Circular RNAs (circRNAs) belong to a class of covalently closed single stranded RNAs that have been implicated in cancer progression. Former investigations showed that hsa-circ-0013561 is abnormally expressed in head and neck squamous cell carcinoma (HNSCC). Nevertheless, the role of hsa-circ-0013561 during the progress of HNSCC still unclear. Methods Present study applied FISH and qRT-PCR to examine hsa-circ-0013561 expression in HNSCC cells and tissue samples. Dual-luciferase reporter assay was employed to identify downstream targets of hsa-circ-0013561. Transwell migration, 5-ethynyl-2′-deoxyuridine incorporation, CCK8 and colony formation assays were utilized to test cell migration and proliferation. A mouse tumor xenograft model was utilized to determine the hsa-circ-0013561 roles in HNSCC progression and metastasis in vivo. Results We found that hsa-circ-0013561 was upregulated in HNSCC tissue samples. hsa-circ-0013561 downregulation inhibited HNSCC cell proliferation and migration to promote apoptosis and G1 cell cycle arrest. The dual-luciferase reporter assay revealed that miR-7-5p and PDK3 are hsa-circ-0013561 downstream targets. PDK3 overexpression or miR-7-5p suppression reversed the hsa-circ-0013561-induced silencing effects on HNSCC cell proliferation and migration. PDK3 overexpression reversed miR-7-5p-induced effects on HNSCC cell proliferation and migration. Conclusion The findings suggest that hsa-circ-0013561 downregulation inhibits HNSCC metastasis and progression through PDK3 expression and miR-7-5p binding modulation.


Background
Head and neck squamous cell carcinoma (HNSCC) is prevalent and highly aggressive, and its incidence continues to rise.It is predicted that by 2030 there will be about 1.08 million new HNSCC cases diagnosed per year [1].Malignant SCC is associated with a grim prognosis, increased mortality rates, and a high propensity for lymph node metastasis [2,3].Despite significant advancements in treatment, the survival rates of HNSCC patients remain disappointingly low [4].Further elucidation of the pathogenic molecular mechanisms is critical for novel therapy developments of HNSCC.
circular RNAs (circRNAs) belong to a particular class of ncRNA molecule that forms through non-canonical splicing mechanisms.Reverse splicing connects 3′ end of an exon to 5′ end of the same or upstream exon, forming a closed loop [5].Former investigations showed that circRNAs like hsa_circ_0016148, hsa_circ_0000264, and hsa_circ_0032822 function importantly in HNSCC progressions [6][7][8].In addition, circRNAs can regulate several cancer-related signaling pathways, like those involving microRNA sponges.Although some functions and underlying mechanisms regarding particular circRNAs participated in HNSCC pathogenesis remain known, further investigation is needed to fully elucidate this biology.
In the present study, we found that hsa-circ-0013561 is upregulated in both HNSCC tissues and cells and that it has a negative positive with the migration and invasion of HNSCC cells.Mechanistically, hsa_circ_0013561 can regulation the malignant progression of HNSCC by miR-7-5p/PDK3 signaling pathway.Revealing hsa_ circ_0013561 as a potential novel effective target for HNSCC treatment.

Tissue samples
Tissue biopsies of carcinoma and paracancerous samples were taken from three HNSCC patients that were pathologically diagnosed at Pathology Department in Shanghai Gongli Hospital from 2021-2022 and had complete clinical data.Biopsies were paraffin-embedded and formalin-fixed, and the paraffin specimen were used for immunohistochemistry. Ethics Committee in Shanghai Gongli Hospital approved the research.Patients provided consents that informed.

Fluorescence in situ hybridization (FISH)
We measured hsa_circ_0013561 expression in tissues using FISH.We used Cy3-bound anti-digoxin and FITCbound anti-biotin antibodies to measure the signal (Geneseed Biotech, Guangzhou, China).We used DAPI to stain nuclei.We used fluorescence Leica microscope to acquire images.
We constructed lentivirus constructs overexpressing hsa_circ_0013561 (sh-circ0013561) and negative control (NC) vectors.Stable Hep-2 knockdown cells were generated after lentivirus infection.After cell passaging, RNA was extracted to verify infection efficiency.

Cell counting kit-8 (CCK8) assay
Following standard protocol, we measured relative cell viability at 1, 2, and 3 days post infection using the CCK-8 (Yeasen, Shanghai, China).We inoculated cells into 96-well plates with 4000 cells per well and five replicates.We next added 10 μl CCK-8 solutions to every well to incubate plates at 37 °C for 1 h in dark.At 0, 24, 48, and 72 h post-seeding, we measured optical density utilizing microplate reader at 450 nm.

5-ethynyl-2′-deoxyuridine (EdU) assay
We measured cell proliferation utilizing EdU assay kit (US EVERBRIGHT, Suzhou, China).Our team inoculated Hep-2 cells transduced with normal control vector as well as Hep-2 cell transductions with sh_circ_0013561 vector into 24-well plates (4 × 10 4 per well) and cultured cells overnight.10 μl of EdU-labeled medium was added to the 24-well plate and then the plate was incubated at 5% CO 2 at 37 °C for two hours.We then fixed plates with 4% paraformaldehyde and 0.5% Triton X-100.We added EdU and DAPI staining solutions to wells and imaged cells with fluorescence microscopy (Leica microscope, Shanghai, China).We calculated EdU incorporation rates as the ratio of total EdU-positive cell (red signal) numbers to DAPI-positive cell (blue signal) numbers.

Colony formation assays
Our team inoculated transduced cells into 6-well plates with density 1000 cells/well in DMEM medium including 10% FBS for one week.Cells were cleaned twice with PBS, fixed with 4% paraformaldehyde for 20 min, and dyed with 0.5% crystal violet for twenty minutes.Colonies comprised of ≥ 50 cells were calculated.

Apoptosis test
We assessed apoptosis using an apoptosis assay (FITC Annexin V Apoptosis Detection Kit I, BD Pharmingen).We first collected non-adherent cells in the culture supernatant.We cleaned adherent cells twice with PBS and trypsinized them for three minutes.We then mixed cells with culture medium to end the digestion, and centrifuged 1 × 10 6 cells at 1000 RPM/min for 5 min.We discarded supernatant, and used 100 μl 1 × binding buffer to resuspend cells and transfer them to Eppendorf tubes.5 μl propidium iodide and 5 μl Annexin V-APC were added to every tube.After mixing, we incubated cells for 15 min at room temperature in dark, followed by adding 500 μl 1 × binding buffer to terminate reactions.Apoptosis was measured utilizing flow cytometry.

Cell cycle assay
Technician measured cell cycle utilizing a cell cycle assay kit (Cell Cycle and Apoptosis Analysis Kit, Beyotime, China).The same method for cell collection for apoptosis detection was used.A total of 500 μl staining buffer, 10 μl RNase A (50 ×) and 25 μl propidium iodide staining solution (20 ×) was used to resuspend cells after washing with PBS.Cells were transferred to Eppendorf tubes, mixed well, and incubated at room temperature.We assessed the cell cycle using a low loading-speed flow cytometry instrument.

Wound healing assay
We inoculated control cells and transduced cells into 6-well plates at 3.5 × 10 5 cells/well to culture them in DMEM medium including 10% FBS.When the confluency reached 90-95%, we created a "scratch" using a 1 ml pipette tip and washed off the detached cells using PBS.Subsequently, serum-free medium was added to culture.At 0, 1, 2 and 3 days post-seeding, we imaged wound area under microscope.Wound area was captured employing Image J software.The cell scratch healing rate was computed as (scratch area at 0 h-scratch area at timepoint)/ scratch area at 0 h × 100%.

Transwell assay
We suspended control and transduced cells in 200 μl serum-free medium with 2 × 10 4 cells/well.We inoculated cells in Transwell plate upper chamber with 8 μm pore size.In addition, we put 600 μl medium including 15% FBS in lower chamber as a chemotactic agent.One day post-seeding, we fixed Transwell membrane utilizing 4% paraformaldehyde, stained it with 0.1% crystal violet, and viewed 3 random view (100 ×) fields with microscope.

Proteomics
We plated Hep-2 cells at 2.0 × 10 6 cells in a 10 cm dish.Cells were incubated at 37 ℃ for 1 d to allow for adherence.We lysed cells using RIPA buffer with 1 mM phenylmethanesulfonyl fluoride.Technician measured protein concentration applying a BCA Kit (Yeasen, China).Protein samples were stored at − 80 ℃.A total of 250 μg protein was incubated with 10 mM DTT at 37 ℃ for one hour, followed by alkylation with 50 mM indole-3-acetic acid at 25 ℃ in the dark for 1 h.Then, our team used 1.5 mL prechilled (− 20 ℃) 100% acetone to clean sediment.The sample was centrifuged at 14,000×g three times for fifteen minutes.Our group diluted samples with 500 μL NH 4 HCO 3 and desalted peptides utilizing SPE C18 cartridges, which we lyophilized under a vacuum prior to nano HPLC-MS/MS analyses.

In vivo experiments
To generate HNSCC mice models, we injected Hep-2 cells (2 × 10 6 ) transduced with either NC or sh-circ0013561 viral vectors into nude mice flank.We measured tumor weights and volumes.After 25 days mice were murder and tumor tissues were isolated for further study.Each group have 6 mice.The Animal Ethics Committee in Shanghai Gongli Hospital approved animal experiments.
For tumor metastasis analyses, luminescence-labeled Hep-2 cells were transduced with sh-circ0013561 or NC.We resuspended 2 × 10 5 cells in PBS and injected them into tail vein for lung metastasis studies, or the pelma for lymphatic metastasis studies.Lung and lymphatic metastases were imaged utilizing in vivo bioluminescence imaging system 4 weeks after injection.We counted metastatic foci in extracted lung tissues based on hematoxylin and eosin (HE) staining.Lymph angiogenesis were detected with CD31 staining.

Statistical analyses
Statistician assessed differences among groups applying an unpaired t-test, and data are expressed as averages.We considered p < 0.05 as statistical significance.Our team performed statistics analysis using GraphPad Prism (GraphPad Inc., San Diego, CA, USA).
Live imaging of Hep-2 cell pulmonary metastasis mouse models demonstrated that hsa_circ_0013561 silencing decreased pulmonary metastases and metastatic foci numbers in lung tissues as assessed with HE staining (Fig. 3G-J).Downregulation of hsa-circ-0013561 resulted in decreased CD31 expression (Fig. 3K-M), suggesting that hsa_circ_0013561 downregulation suppressed HNSCC cell invasion.

Discussion
Malignant squamous cell carcinoma, particularly in the case of HNSCC, is characterized by a grim prognosis, significant lymph node metastasis, and elevated mortality rates [9,10].The development of numerous human tumors relies heavily on complex signaling networks, which are intricately regulated by various non-coding RNAs such as circRNAs [8,11].The specific involvement Proteomics and bioinformatics data demonstrated that PDK3 and miR-7-5p are downstream hsa-circ-0013561 targets, which was further validated using a luciferase reporter assay.hsa-circ-0013561 downregulation enhanced miR-7-5p expression.Altered miR-7-5p expression was involved in the tumorigenic processes, which is supported by previous reports that miR-7-5p may act as tumor inhibitor in NSCLC, colorectal cancer and pancreatic ductal adenocarcinoma [12][13][14][15].Our data verified that miR-7-5p downregulation reversed sh-circ-0013561 inhibitory effects upon HNSCC proliferation and migration, suggesting that hsa-circ-0013561 silencing inhibits HNSCC progression by enhancing miR-7-5p expression.
In summary, our data supports that hsa-circ-0013561 downregulation suppresses HNSCC proliferation via miR-7-5p/PDK3 signaling axis.The data endorses hsacirc-0013561 as a candidate diagnostic biomarker, which is a promising therapy target.

Fig. 1
Fig. 1 Downregulation of hsa-circ-0013561 inhibits HNSCC cell proliferation and tumor growth.A, B FISH detection showing the expression and subcellular distribution of hsa-circ-0013561.C qRT-PCR detection showing the expression of hsa-circ-0013561 in Hep-2 cells after transduction with NC or sh-circ0013561.*** p < 0.001 vs. si-NC.D CCK-8 detection showing Hep-2 cell proliferation.*** p < 0.001 vs. NC.E, F EdU assay showing Hep-2 proliferation after transduction with NC or sh-circ0013561.** p < 0.01 vs. NC.G, H Colony formation assay showing Hep-2 proliferation after transduction with NC or sh-circ0013561.** p < 0.01 vs. NC.All data are expressed as the mean ± SD.I Representative images of HNSCC tumor formation in nude mouse xenograft models.J Summary of mouse tumor volumes measured every three days.***p < 0.001 vs. sh-NC.K, L Tumor volumes and weights measured 25 days post-injection.**p < 0.01, ***p < 0.001 vs. NC.M, N Immunohistochemistry showing the percentage of Ki67-positive cells.***p < 0.001 vs. NC.All data are presented as the mean ± SD.All experiments were repeated three times